When cultured in polystyrene dishes subjected to previous treatment and supplied with a serum-containing medium, hog thyroid cells form monolayers displaying dome-like arrangements after three to four days. Cells involved in formation of "domes" are morphologically polarized; the apical microvilli of these cells point toward the culture medium. When the tissue is cultured in untreated polystyrene dishes, thyroid cells remain in suspension; their aggregates swell progressively and form hollow spheres encompassed by a single layer of cells. The polarity of the cells forming such spheres is inverse in comparison to the condition characteristic of the intact thyroid gland. When culture medium is supplemented with TSH, PGE1, PGE2 or dBC, structures resembling true follicles are formed in both types of cultures. Gelatin, added to suspension cultures, is also capable of promoting follicle formation. Cultured thyroid cells regularly form an epithelial layer as a result of the interaction of cellular processes. However, the polarization of this layer depends on culture conditions. Thus, structures with either a normal follicle-like polarization of their cells or showing an inverted type of polarization can be obtained.
Two different independent processes are operating in cultured thyroid cells to regulate adenylate cyclase/cyclic AMP responsiveness to thyroid stimulators (thyrotropin and prostaglandin Ez) :firstly, refractoriness or negative regulation [preceding paper], which is specific for each thyroid stimulator, is not mediated by cyclic AMP and is not accompanied by alteration of adenylate cyclase activity; secondly, positive regulation which is characterized by an augmentation of the cyclic AMP response stimulated by thyrotropin and prostaglandin E2. This process is not specific for each thyroid stimulator and is a state of increased susceptibility of cyclic AMP synthesis to stimulation, accompanied by increased activity of the catalytic subunit of adenylate cyclase. Positive regulation is apparently mediated by increased intracellular cyclic AMP levels. It is a time-dependent and dose-dependent process. Very low concentrations ( 5 -50 pU/ml) of thyrotropin augmented cyclic AMP synthesis stimulated by thyrotropin and prostaglandin E2 whereas higher concentrations (above 0.1 mU/ml) augmented prostaglandin El stimulation but induced refractoriness to thyrotropin. Prostaglandin E2 (0.1 to 10 pM) augmented thyrotropin stimulation and dibutyryl adenosine 3' : 5'-monophosphate (0.3 to 2 mM) augmented thyrotropin and prostaglandin Ez stimulation. Positive regulation is a slow process which develops within days and increases up to day 5 in culture. Experiments using inhibitors suggested that protein synthesis is required for the full expression of the increase in adenylate cyclase activity induced by the studied thyroid stimulators.We have recently shown that the cyclic AMP response to thyrotropin of thyroid cells in culture was under a dual regulation by thyrotropin : chronic exposure of the cells to physiological concentrations of the hormone augmented the subsequent cyclic AMP response to thyrotropin (positive regulation), whereas moderate to high concentrations of thyrotropin induced refractoriness to further thyrotropin stimulation [l].The conditions for the development of refractoriness to thyrotropin and prostaglandin Ez have been described in a preceding paper [2]. In this report, we analyze the mechanism of the positive regulation of the acute cyclic AMP response to thyrotropin and prostaglandin E2 as influenced by chronic treatment of cultured thyroid cells with these stimulators. MATERIALS AND METHODSThe methods and the materials used have been described in the preceding paper [ 2 ] . Cells cultured in the presence of thyrotropin (10 mU/ml), prostaglandin EZ (1 pM) or dibutyryl cyclic AMP (0.4 mM) will be referred to further as thyrotropin-treated, prostaglandin E2-treated or BtzcAMP-treated cells, respectively. Cells cultured in the absence of these compounds will be referred to as control cells.Cyclic AMP synthesis using prelabeling technique with 14C-labeled adenine [3] was measured as reported previously [4] with minor modifications. Duration of preincubation with [14C]adenine was 40 rnin. To preserve cell viab...
Isolated porcine thyroid cells, cultured in the presence of thyrotropin ( 2 0.25 mU/ml) or prostaglandin EZ ( 2 0.1 pM), showed decreased adenosine 3': 5'-monophosphate (cyclic AMP) response to further thyrotropin or prostaglandin EZ stimulation, respectively.Kinetics of the refractory process to thyrotropin and prostaglandin EZ are different : ( a) maximal refractoriness to prostaglandin EZ was attained after 2 -6 h exposure to prostaglandin EZ while refractoriness to thyrotropin was maximal only after 12-24 h ; (b) the degree of refractoriness to prostaglandin Ez was much greater than that to thyrotropin.Refractoriness to thyrotropin or prostaglandin EZ is characterized : by specificity for each thyroid stimulator; by dependence upon the dose of thyrotropin or prostaglandin E2 in culture, e.g. induction of high degree of refractoriness with 0.5 mU/ml thyrotropin (or 1 pM prostaglandin Ez), which elicits only a small cyclic AMP increase; by time requirement for induction; by partial effect; by changes of maximum activation of cyclic AMP response; by reversibility.This refractoriness of the cyclic AMP response was not induced by dibutyryl adenosine 3': 5'-monophosphate. It was not attributed to increased cyclic AMP-phosphodiesterase activity, but to alterations in the receptor-adenylate cyclase system. Prevention of refractoriness to thyrotropin or prostaglandin EZ by incubation of cells in the presence of actinomycin D, puromycin and cycloheximide suggests that new RNA and protein syntheses are required for the development of the refractory state.
Summary -Nueleophilie primary amino groups of bovine~-Iaetoglobulin B were modified with different aldehydes by an addition-elimination reaction eommonlynamed ."reductive alkylation". Subsequently resulting imines (Sehiff bases) were redueed, produeing secondary amines derivatives of this protein. Two kinds of ligands, differing by their hydrophobieity, were coupled to this protein: 81% and 69% of~-Iaetoglobulin amino groups were modified with glucose and maltose, respectively; 31% and 44% of~-Iaetoglobulin amino groups were modified with vanillin and benzaldehyde, respectively. Reversed-phase HPLC and eleetrophoresis of modified~-Iactoglobulin indicated that, in the case of glycosylation, the condensation was rather homogeneous, while the modification with aromatie substituents was quite heterogeneous.Water solubility of'glycosylated~·Iactoglobulins did not differ mueh from that of 13-lactoglobulin, on the whole pH range. By contrast, aromatie derivatives exhibited a very low solubility near the isoelectrie point. Emulsifying activity and emulsion stability were also eheeked on the whole pH range. Glucose-modified 13-lactoglobulin had improved emulsifying properties whatever the pH, as compared to native protein. Emulsifying activity of aromatie derivatives was improved near the isoelectrie point. Maltosylated-, vanillin-modifiedand benzaldehydemodified-13-laetoglobulin exhibited higher emulsion stability in the acidie pH region but lower emulsion stability in the alkaline pH range, as compared to native 13-lactoglobulin. Lee et al (1979) have used the reductive alkylation reaction described by Means and Feeney (1968) for the coupling of reducting oligosaccharides to casein with sodium cyanoborohydride in aqueous solution and checked the nutritive value of these substituted proteins. Recently, Courthaudon et al (1989) used the reductive alkylation procedure to bind different carbohydrates on Iysyl residues of bovine whole casein and described the effects on solubility and viscosity of this protein.f3-lactoglobulin is a well-characterized protein (McKenzie, 1971;Creamer et al, 1983;Sawyer et al, 1985;Papiz et al, 1986) which exhibits good solubility but relatively weak emulsifying properties. The aim of our study was to apply reductive alkylation on f3-lactoglobulin in order to bind either reducing sugars (glucose and maltose) or hydrophobic aldehydes (benzaldehyde and vanillin) and to determine the comparative effects of these ligands on solubility and emulsifying properties of 13-lactoglobulin.
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